When serum induces growth of quiescent cultured cells, the earliest detectable event is increased ion flow across the plasma membrane. I propose to study the relationship between ion flow and cell growth. The earliest detectable change in quiescent cells stimulated to grow by serum, purified growth factors, or the phorbol diester tumor promoter 12-tetradecanoyl-phorbol-13-acetate (TPA) is an increase in ouabain-inhibitable potassium uptake which results in an increase in cellular K ion content. I have confirmed that ouabain also inhibits cell growth, and have shown that this inhibition occurs in the early and mid-G0/G1 phase of the cell cycle and results from a specific effect on the cell membrane Na-K ions pump. However, ouabain may inhibit growth by inhibiting protein synthesis. I propose to determine whether the increase in potassium uptake is necessary for growth. Serum has been fractionated into two sets of growth factors which regulate different events in the G0/G1 phase of the Balb/c-3T3 cell cycle. A platelet-derived growth factor (PDGF), recently purified to homogeneity in the laboratory with which I am associated, is released into serum during the clotting process. A short treatment with PDGF induces cells to become competent to synthesize DNA. Competent cells do not synthesize DNA unless continuously treated with platelet poor plasma. One of the critical hormones in plasma, which regulates a distinct event later in the cell cycle, is somatomedin, a family of growth regulatory hormones with insulin-like activity. The small DNA tumor virus SV40 overrides the cell's requirement for both the PDGF and plasma components of serum. I have shown that the tumor promoter TPA partially replaces either PDGF or plasma, but not both. I have shown that ouabain inhibits plasma growth factor-mediated progression through the cell cycle, but has no effect on the initiation of cell cycle by PDGF or TPA. I will study the roles of the purified growth factors (PDGF and somatomedin), TPA, and SV40 infection in the direct control of plasma membrane ion flux.Na ion and K ion flux will be measured in intact cells and in membrane vesicles. A systematic analysis of direct effects of the agents on membrane ion flux will be performed using membrane vesicles.